Cellular mediators of innate immunity do not express rearranged V regions, yet have the capacity to recognize and respond to target cells in the absence of antibody. Recently an ancient family of pathogen recognition receptors, the toll like receptors (TLRs) was discovered in humans and mice and two of these, TLRs2 and 4 were shown to trigger inflammatory responses to LPS and other pathogen derived substances. We hypothesized that pathogen recognition by iDC is mediated by toll like receptors (TLRs), and asked which TLRs are expressed during the progression of monocytes to mDC. We first measured mRNA levels for TLRs 1-5 and MD2 (a protein required for TLR4 function) by Northern analysis. For most TLRs, message expression decreased several fold as monocytes differentiated into iDC, but opposing this trend, TLR3 and MD2 showed marked increases during iDC formation. When iDC were induced to mature with LPS or TNF-a, expression of most TLRs transiently increased and then nearly disappeared. Stimulation of iDC, but not mDC, with LPS resulted in the activation of IRAK, an early component in the TLR signalling pathway, strongly sugesting that LPS signals through a TLR. Surface expression of TLRs 1 and 4, as measured by mAb binding, was very low, corresponding to a few thousand molecules per cell in monocytes, and a few hundred or less in iDC. Thus, TLRs are expressed in iDC and are involved in responses to at least one pathogen derived substance, LPS. Human microvascular endothelial cells respond to LPS through TLR4 and we have found that TLR1 inhibits this response. When transfected into 293 cells, TLR1 binds to other TLRs, including TLRs 2, 4 and 5, and preliminary results using modified TLR1 suggest that binding occurs through the extracellular domain. TLR1 is the most ubiquitously expressed TLR, and our data suggest that it serves a function in negatively controlling TLR responses to pathogens. Previously we showed that tumor induced immunosuppression was mediated by a subset of myeloid cells that migrate to lymphoid organs and block T and B cell responses to antigen. These myeloid suppressor cells (MSC) have been immortalized, retain their suppressive functions, and provide a source of cells for studying mechanisms of suppression. While MSC strongly block T cell proliferation in response to stimulation by antigen, allogeneic cells and Con A, we have observed that two different mechanisms are used to block allogeneic and mitogen driven responses. Suppression of allogeneic responses was NO independent, accompanied by a loss in IL-2 secretion by the responder cells, and was irreversible after 24 hr contact with MSC, consistent with cell death within the responder population. By contrast, inhibition of mitogenic and peptide specific responses was reversible, did not entail a loss in IL-2 production, and required NO and IFN-g. A unique capacity of MSC lines to produce high levels of NO in response to IFN-g and a contact-dependent signal from the responder population distinguished them from phenotypically related non-suppressive counterparts. Western blots for phospho-STAT5, Erk, and Akt demonstrated that MSC treatment directly impaired the IL-2 signaling pathway in mitogen-stimulated T cells, since none of these molecules was phosphorylated in response to IL-2 after incubation with the MSC. Recent results suggest that suppression may operate on other pathways as well, for example, we have found that NO blocks IL-3 and IL-12 pathways in addition to IL-2. We are currently testing the hypothesis that all Janus kinases are susceptible to inhibition by NO, and that as a result, MSC would have the capacity to inhibit most cytokine-induced responses.